Elevated ozone disrupts mating boundaries in drosophilid flies

Animals employ different strategies to establish mating boundaries between closely related species, with sex pheromones often playing a crucial role in identifying conspecific mates. Many of these pheromones have carbon-carbon double bonds, making them vulnerable to oxidation by certain atmospheric oxidant pollutants, including ozone. Here, we investigate whether increased ozone compromises species boundaries in drosophilid flies. We show that short-term exposure to increased levels of ozone degrades pheromones of Drosophila melanogaster, D. simulans, D. mauritiana, as well as D. sechellia, and induces hybridization between some of these species. As many of the resulting hybrids are sterile, this could result in local population declines. However, hybridization between D. simulans and D. mauritiana as well as D. simulans and D. sechellia results in fertile hybrids, of which some female hybrids are even more attractive to the males of the parental species. Our experimental findings indicate that ozone pollution could potentially induce breakdown of species boundaries in insects.


Supplementary Discussion
Reproductive isolation i.e. the lack of gene flow between populations is regarded as an important driver of speciation (reviewed by Mallet, 2006)  1 .Such reproductive isolation often is a result of geographic isolation of so-called allopatric populations that via different selective pressures or genetic drift become more and more dissimilar and finally speciate.
In addition, few examples of sympatric speciation (i.e. the evolution of a new species in close proximity of its ancestral species) have been identified in e.g.African cichlids [2][3][4] or the apple maggot fly [5][6][7] .Finally, some species seem to be the result of hybrid speciation 8 , where the hybridization between closely related species finally results in the evolution of a new species.The most prominent insect example is the species-rich genus of Heliconius butterflies 9 , where hybridization of two closely related species can result in a fertile hybrid that by its wing pattern and behavior is reproductively isolated from the two donor species 10 .Similarly, there is one reported case of hybrid speciation for Drosophila, where hybrids of D. ananassae and D. parapallidosa obviously evolved into the new species D. cf.parapallidosa 11 .
Our manuscript deals with four species of the Drosophila melanogaster complex, because both their pheromone blends and their sexual behavior are well established.D. sechellia, and D. mauritiana most probably have evolved from a large mainland population of a shared ancestor with D. simulans through allopatric speciation based on two island colonization events 12 .D. simulans, like D. melanogaster nowadays is globally distributed and also occurs on the Mauritius and the Seychelles, i.e. the islands originally inhabited by D. mauritiana and D. sechellia.It has been shown that in Drosophila flies during speciation usually first prezygotic isolation (i.e. via courtship and mating boundaries) and afterward postzygotic isolation (via hybrid sterility and inviability) are established 13,14 .D. simulans, D. sechellia, and D. mauritiana belong to the simulans species complex and have established prezygotic isolation based on e.g.species-specific pheromonal blends [15][16][17][18] and courtship songs [19][20][21] .Their post-zygotic isolation, however, is incomplete, as only male hybrids are sterile, while female hybrids are fertile.On both islands, gene flow via hybridization events between D. simulans and its close relatives has been reported (with D. sechellia 22 ; with D. mauritiana 23 ), suggesting that presynaptic isolation between these species is not absolute.Our data reveal, that oxidant pollutants like ozone have the potential to corrupt prezygotic isolation and, hence, make hybridization events more likely.As at least some of the resulting hybrids seem to be competitive regarding mate choice (Fig. 3) and reproduction (Fig. S6), such hybridization events potentially could result in ongoing gene flow between sympatric species.As for these species several genetic incompatibilities have been reported [24][25][26][27][28] , it, however, is questionable, whether ongoing gene flow in this species complex has the potential to finally result in hybrid speciation.In addition, within the Drosophila genus, however, many more sympatric species pairs exist that can hybridize 29 and whose species boundaries therefore might also become affected by increased levels of ozone.Obviously other parameters (e.g. the females' acceptance of the male song) play an additional roles here.

Figure S1 .
Figure S1.Quantitative analysis of cVA and pheromonal CHCs after ozone exposure recovery in four Drosophila species.a, Time line of experiment.Ozonated and control flies are exposed for two hours to 100 ppb ozone and ambient air, respectively.After that flies were placed into food vials and we let them recover for 24h or 48h.b, Quantitative analysis after 24h recovery.c, Quantitative analysis after 48h recovery.The box plots present median values and quartiles, whiskers the minimum and maximum values, and dots the individual data points.Two-sides Unpaired t-test.*p<0.05;**p<0.01;***p<0.001;NS, no significant difference.Because of the GC-MS components e.g.ion source, column, and the concentration of internal standards are varying, hence we only compare our results from the same test sequence to minimize the variations.

Figure S2 .
Figure S2.Mating frequency during 6 hours when a female can choose between two conspecific males.The numbers in the donut plots indicate the experiments that resulted in single mating (colored) or no mating (white).We never observed that a female mated twice during these 6 hours.D. sim: D. simulans; D. sec: D. sechellia; D. mau: D. mauritiana.

Figure S3 .
Figure S3.A hybridization overview between four Drosophila species and male posterior lobe morphology of Drosophila purebred species and hybrids.a, Hybridizations between four Drosophila species.Black letters, hybrids obtained in this study; gray letters, hybrids reported by previous references.b, Morphology of male posterior lobes.D. mel: D. melanogaster; D. sim: D. simulans; D. sec: D. sechellia; D. mau: D. mauritiana.All hybrids are F1 and named as F0 female × F0 male, e.g.D. sim-mel is a hybrid offspring of a female D. sim and a male D. mel.Rep. indicate replicates 1-3.

Figure S4 .
Figure S4.Ozone expose to 50ppb ozone is not enough to induce hybridization among closely related Drosophila species.Individual female flies are confronted with one intraand one interspecific male for six hours.The existence or absence of hybrid offspring informs about the succeeding male.Donut plots of success rates of ozonated (middle) and control (bottom) conspecific and allospecific males courting D. melanogaster and D. simulans.Sample sizes are provided in donut centers.Numbers in segments depict numbers of successful males.White segments, no male mated the female.Two-tailed Fisher's exact test.

Figure S5 .
Figure S5.Pheromone quantitative analysis of D. mel, D. sim, D. mau, D. sec, and their hybrids.a and ai, pheromone of D. mel and hybrids.b and bi, pheromone of D. mau and hybrids.c and ci, pheromone of D. sim and hybrids.d and di, pheromone of D. sec and hybrids.All hybrids are F1 and named as F0 female × F0 male.Fly names in bold characters indicate mating preference in competitive mating assays (see Fig.3).The box plots present median values and quartiles, whiskers the minimum and maximum values, and dots the individual data points.One-way ANOVA with Tukey's multiple comparisons test for hybrid of D. sim-mau, D. mel-sec, D. sim-mel, D. sec-mel, and male D. sim-sec.While t-test for hybrid of D. mel-mau, D. mel-sim, and female D. sim-sec.NS indicate no significant

Figure S6 .
Figure S6.Fitness of female hybrids and purebred flies regarding egg numbers, hatching rates, development time, and the survival rate from egg to adult.a, Egg numbers of each female during 5 days after mating.Figure shows mean ± SD. b, Egg hatching rate after 48h.c, Development time (days) from egg to pupa. Figure shows mean ± SD. d, survival rate from egg to adult.The x-axis shows the parental combination (female/male).Kruskal Wallis with Tukey Kramer post-hoc test for selected pairs for a and c.Chi-square test with Bonferroni adjustment for b and d.Stars or characters with orange, green, and brown depict the comparison with D. sim, D. sec.and D. mau, respectively.*p<0.05;**p<0.01;***p<0.001;NS, no significant difference.